Billions of cardiomyocytes die after myocardial infarction and fail to be replaced, reducing pumping function and causing heart failure - the leading global cause of death. Understanding how cardiomyocyte proliferation is regulated remains central for determining mechanisms of heart regeneration and effectively targeting therapies to address heart failure. Previous studies have demonstrated the non-DNA binding homeodomain protein, HOPX, governs cardiomyocyte proliferation during heart development. We aimed to determine the role of HOPX in adult heart regeneration. Utilizing zebrafish, a model organism with innate heart regeneration, we tested the role of hopx in regulating cardiomyocyte proliferation at the organ-level in vivo. We generated cardiomyocyte-specific hopx-overexpressing zebrafish and assessed their regenerative response seven days after ventricular injury. Wound-adjacent cardiomyocytes from hopx-overexpressing zebrafish proliferated significantly less compared to clutchmate controls, demonstrating ectopic hopx expression is sufficient to blunt acute cardiac regeneration. To determine if HOPX deficiency promotes proliferation in human cardiomyocytes in vitro, we generated cardiac organoids using CRISPRi HOPX conditional loss-of-function human induced pluripotent stem cells (hiPSCs). HOPX knockdown (KD) tissues reflecting the metabolic environment of early heart development produced significantly increased force attributable to increased cardiomyocyte proliferation relative to control tissues. To investigate the transcriptomic signature of HOPX loss-of-function, we performed genome-wide CAGE-sequencing. HOPX KD resulted in significant enrichment for cholesterol biosynthesis, an essential regulatory pathway, among others, underlying cardiomyocyte proliferation. To test the mitogenic potential of HOPX deficiency in post-mitotic cardiomyocytes, we metabolically-matured hiPSC-derived cardiac tissues. We show metabolic maturation is sufficient to block HOPX-dependent cardiomyocyte proliferation, likewise resulting in no significant difference in force generation compared to controls. Collectively, this study provides critical insights into regulatory mechanisms underlying heart development and regeneration by demonstrating that HOPX directly regulates cardiomyocyte proliferation after organ-level injury and establishing that the mitogenic potency of HOPX is dependent on the cardiomyocyte metabolic state.